Two protein kinases have been identified that have the capacity to affect translational control of protein synthesis by phosphorylation of the alpha subunit of peptide initiation factor 2, eIF-2. One is induced by interferon and activated by double-stranded RNA. A different kinase is activated by heme deficiency in reticulocytes. Both kinases phosphorylate the same site in a 4 kilodalton, terminal segment of eIF-2-alpha with concomitant inhibition of protein synthesis, thereby exerting control through a common mechanism. A crucial question for understanding translational control involves the mechanism by which these kinases are activated and inactivated. Phosphorylation of both kinases appears to be an essential step in their activation. Potentially, the state of phosphorylation of either eIF-2 or the kinases can be regulated by a cognate phosphatase. A 55 kilodalton, Mn2+-dependent protein phosphatase has been purified to homogeneity fromreticulocytes. Its activity is modulated by a 230 kilodalton protein that appears to be associated with spectrin in the cytoskeleton of reticulocytes. We have named this protein "regulin." It is extremely sensitive to proteolysis, and at least some of the larger peptides derived from it are active in regulating the phosphatase. The interaction of regulin and its peptides with thephosphatase has been studied using fluorescence techniques after labeling of the phosphatase with a maleidyl derivative of coumarin. Mn2+ is required for efficient interaction of regulin with the phosphatase, and it appears that both Mn2+ and regulin cause conformational changes in the enzyme. Equilibrium dialysis experiments have shown that 12 Mn2+ are bound per phosphatase witha Kb of 0.5 mM-1. Co2+, Fe2+, and Zn2+ bind to the protein and inhibit the Mn2+-activatedenzymatic activity apparently by competition with Mn2+. The Kb for Zn2+ is 9 micromolar. (F)